This invention relates to a novel method of inhibiting hepatitis B virus and, more particularly, to the use of N-alkyl derivatives of 1,5-dideoxy-1,5-imino-D-glucitol for inhibiting replication and secretion of hepatitis B virus in cells infected with said virus.
Hepatitis B Virus (HBV) is a causative agent of acute and chronic liver disease [Ayoola et al., Bull. World Health Organ. 66, 443-455 (1988)]. Although effective vaccination is available [two HBV vaccines currently available are Merck's Recombivax HB and SmithKline Beecham's Engerix-B], there are still more than 300 million people worldwide chronically infected with the virus [Eder et al., in Progress in Liver Diseases, eds. Popper and Schaffner (Grune & Stratton, Orlando, FL), vol. 8, pp. 367-394 (1986)]. For them, the vaccine has no therapeutic value. According to Dr. Richard Duma, executive director of the National Foundation for Infectious Diseases, an estimated 300,000 cases of HBV infection occur annually in the United States alone [Med. World News 34(8), 20-21 (1993)]. Between 25 to 40% of those who are chronically infected with HBV develop serious liver disease. It is therefore important to find effective anti-HBV therapies.
Alpha interferon has been used for treatment of HBV infection with promising results in some patients [Hoofnagle and Jones, Seminars in Liver Disease 9, 231-233 (1989); and Perrillo, Seminars in Liver Disease 9, 240-248 (1989)]. The only treatment for chronic HBV infection currently approved by the U.S. FDA is recombinant interferon alfa-2b (Intron A, Schering-Plough). Clinical tests on the use of the nucleoside analog, fialuridine, for treatment of chronic hepatitis B were suspended recently due to drug-related liver failure in six of 20 patients. Consequently, there is a great need for a safe drug treatment of hepatitis B.
Recent reports suggest that the virus encoded DNA polymerase, which functions as a reverse transcriptase, is an attractive target [Doong et al., Proc. Natl. Acad. Sci. USA 88, 8495-8499 (1991); Lee et al., Antimicrob. Aaent Chem. 33, 336-339 (1989); Price et al., Proc. Natl. Acad. Sci. USA 86, 8541-8544 (1989); and Venkateswaran et al., Proc. Natl. Acad. Sci. USA 84, 274-278 (1987)].
Other virus-mediated processes have not been targeted for anti-viral intervention. Effective antiviral therapy for HBV is likely to involve multiple strategies, including agents that influence the host immune system as well as those that interfere with different steps in the life cycle of the virus. It is therefore of interest to explore the possibility that other, non-polymerase mediated steps in the virus life cycle are vulnerable to intervention.
1,5-Dideoxy-1,5-imino-D-glucitol (which is also known as 1-deoxynojirimycin or DNJ) and its N-alkyl derivatives are known inhibitors of the N-linked oligosaccharide processing enzymes, .alpha.-glucosidase I and II. Saunier et al., J. Biol. Chem. 257, 14155-14161 (1982); Elbein, Ann. Rev. Biochem. 56, 497-534 (1987). As glucose analogs they also have potential to inhibit glucosyl-transferases. Newbrun et al., Arch. Oral Biol. 28, 516-536 (1983); Wang et al., Tetrahedron Lett. 34, 403-406 (1993). Their inhibitory activity against the glucosidases has led to the development of these compounds as antihyperglycemic agents and antiviral agents. See, e.g., PCT Int'l. Appln. WO 87/03903 and U.S. Pat. Nos.: 4,065,562; 4,182,767; 4,533,668; 4,639,436; 4,849,430; 4,957,926; 5,011,829; and 5,030,638.
Studies on the effect of inhibitory agents on hepatitis B virus (HBV) have been sparse heretofore due to the lack of permissive cell culture systems for assay purposes. That is, the inability heretofore to reproduce and productively infect tissue cultures with the virus has been a serious limitation to the discovery of useful anti-HBV agents.
In one study, N-methyl deoxynojirimycin has been reported to inhibit the formation of mouse hepatitis virus (MHV) whereby the appearance of E2 on the cell surface is delayed. See Repp et al., J. Biol. Chem. 280, 15873-15879 (1985); Datema et al., Pharmac. Ther. 33, 221-286, at 260 (1987). However, MHV is unrelated to the hepatitis B virus (HBV). On the one hand, HBV is a member of the Hepadnavirus family and is a small virus pathogen in humans. The HBV size is approximately 42 nM with a DNA genome size of 3.5 kb.
On the other hand, MHV is a member of the Coronavirus family, and is a large RNA-containing virus that is not pathogenic to humans, although human coronavirus pathogens that cause upper respiratory tract infections are common. The MHV size is about 100-150 nM (being rather pleiotropic), with an RNA genome size of approximately 30 kb. There are very few similarities between HBV and MHV. Further background information and a complete description of Coronaviruses (including MHV) can be had by reference to K. Holmes, in Virology, 2d edition, ed. by B. Fields, pp. 841-856, Raven Press, New York, N.Y., 1990.
The inability to predict the results from one virus to another is evident from the recent reports by two different scientific groups that hepatitis delta virus (HDV) secretion was not dependent upon HBV sAg glycosylation. W. Hui-Lin et al., Abstr. 115, and C. Gureau et al., Abstr. 117, in Abstracts of Papers Presented at the 1994 Meeting, "Molecular Biology of Hepatitis B Viruses," Oct. 3-6, 1994, Institut Pasteur, Paris, France.
HDV does not specify its own envelope protein. It infects the same cells as HBV, and uses the HBV S antigen (HBV envelope protein) to make the infectious, mature HDV particle. By way of distinction, HBV secretion is dependent upon glycosylation and glycan trimming. That is, although HBV and HDV are composed of the same envelope proteins, HDV secretion is glycosylation independent whereas HBV is very sensitive to glycosylation.
The effect of the glycosylation inhibitor, tunicamycin, on hepatitis B virus cell culture systems has been described by Pizer et al., J. Virol. 34, 134-153 (1980); Datema et al., supra at 270. However, tunicamycin undesirably and completely prevents the addition of N-linked oligosaccharides to newly synthesized polypeptide. That is, treatment with tunicamycin results in complete inhibition of N-linked glycosylation of proteins and is very toxic to cells. Moreover, tunicamycin treatment of HBV infected cells resulted in no significant reduction of HBV secretion.